Recruitment of Distinct Chromatin-modifying Complexes by Tamoxifen-complexed Estrogen Receptor at Natural Target Gene Promoters in Vivo

Xue-Feng Liu,Milan K Bagchi

doi:10.1074/jbc.m311932200

Abstract

Tamoxifen, a breast cancer therapeutic, is a tissue-selective estrogen receptor modulator (SERM), which acts as an antiestrogen in the mammary tissue and displays estrogenic activity in other tissues such as bone and uterus. In order to understand the mechanisms underlying the antiestrogenic effect of this prototype SERM, we performed an analysis of the cofactors that interact with ER complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7. Employing chromatin immunoprecipitation (ChIP), we observed that treatment with OHT rapidly induces the binding of ERalpha to the E-responsive promoter regions of pS2 and c-myc genes. Promoter-bound OHT-complexed ERa coordinately recruited the components of a multiprotein complex containing the corepressor NCoR, histone deacetylase 3 (HDAC3), and a WD40-repeat protein TBL1. Surprisingly, the OHT-complexed ERalpha also recruited a chromatin-remodeling NuRD complex in which histone deacetylase 1 (HDAC1) is associated with several polypeptides including metastasis-associated protein 1/2 (MTA1/2), and SWI2/SNF2-related ATPase Mi2. Kinetic studies revealed that following OHT addition the recruitment of these HDAC complexes to pS2 or the c-myc promoter occurs in a sequential manner; the NCoR-HDAC3 complex is recruited earlier than the NuRD complex. Serial ChIP experiments indicated that the ER-NCoR-HDAC3 and ER-NuRD complexes are distinct, and they do not occupy the target gene promoter simultaneously. We also established a close temporal link between the appearance of the HDAC complexes at the E-responsive regions of pS2 and c-myc promoters, local hypoacetylation of specific lysine residues in N-terminal tails of histones H3 and H4, and disappearance of RNA polymerase II from the target gene loci. Collectively, our studies indicated that transcriptional repression by tamoxifen-bound ER at E-regulated gene promoters involves a dynamic interplay of multiple distinct chromatin-modifying/remodeling complexes.

Highlights

The steroid hormone estrogen (E)1 is a key regulator of growth and development of normal breast tissue [1, 2]
In order to understand the mechanisms underlying the antiestrogenic effect of this prototype selective estrogen receptor modulator (SERM), we performed an analysis of the cofactors that interact with E receptors (ER) complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7
We embarked on an analysis of the nature of the corepressor complexes recruited by tamoxifencomplexed ER at the promoters of E-responsive genes, pS2 and HAT, histone acetyltransferase; ChIP, chromatin immunoprecipitation assay; GST, glutathione S-transferase; histone deacetylases (HDACs), histone deacetylase; OHT, 4-hydroxytamoxifen; SERM, tissue-selective estrogen receptor modulator

Summary

Introduction

The steroid hormone estrogen (E) is a key regulator of growth and development of normal breast tissue [1, 2]. The recruitment of a coactivator complex containing HAT activity by a promoter-bound steroid receptor is thought to lead to local hyperacetylation of lysine-rich tails of histones H3 and H4, which may decondense nucleosomes and facilitate the binding of transcription factors to the underlying DNA regulatory elements, leading to gene activation [9, 10]. Previous in vitro and in vivo studies indicated that ER bound to tamoxifen interacts with the corepressor NCoR or SMRT, which are known to associate with histone deacetylases (HDACs) [13,14,15,16,17]. We embarked on an analysis of the nature of the corepressor complexes recruited by tamoxifencomplexed ER at the promoters of E-responsive genes, pS2 and HAT, histone acetyltransferase; ChIP, chromatin immunoprecipitation assay; GST, glutathione S-transferase; HDAC, histone deacetylase; OHT, 4-hydroxytamoxifen; SERM, tissue-selective estrogen receptor modulator. Most importantly, during tamoxifen-induced suppression of ER-mediated gene transcription, the time course of recruitment of the HDAC complexes precisely coincided with that of deacetylation of histone H3 and H4 tails at the target promoters, providing critical support for the hypothesis that tamoxifen functions as an antagonist in the breast cells by inducing chromatin modification

Methods

Results

Conclusion